The invention relates to the provision of polyacrylamide gel filled capillaries for electrophoresis of improved performance and suitably are provided by using a special process of production. After special steps of surface pretreatment the gels polymerized in such capillaries are stable for long series of routine separations. The formation of bubbles during the formation of the gel within the capillary is avoided. Such bubbles affect the usefulness of the capillaries. Separations of various oligonucleotide samples illustrate the performance of the novel gel capillaries. The symmetry of peaks as achieved in such separations is superior to that obtained without the novel surface pretreatment.
For analytical applications, the classical electrophoretic separations using slab gels have certain advantages in comparison to gel capillaries. Several samples can be separated side by side simultaneously and with the same separation parameters. Such parallel separations allow accurate matching of the position of individual components in the eltropherogram on the slab which may originate from different samples. Separations in gel capillaries can be performed with higher resolution or speed of analysis, however.
Therefore and for other reasons the further miniaturization of gel electrophoretic systems has gained in interest, recently. Capillaries with small internal diameters are characterized by a larger surface-area-to-volume ratio, which is of advantage for better dissipation of Joule heat generated during electrophoretic runs at higher field strength. The major problems of analytical application of CGE are related to the still insufficient sensitivity of detection of the separated species and the necessity of the generation of various types of gels inside the capillary. The unavoidable shrinking which accompanies the generation of the gel by polymerization and crosslinking within a narrow bore capillary leads to inhomogeneities of the filling. Furthermore the .zeta.-potential which builds up on the fused-silica surface underneath the gel may extrude a small section of the gel out of the capillary at the cathodic end. Problems of this kind are avoided by a special sequence of surface pretreatment procedures.
An important step of surface modification procedure to be described has been applied before in free zone electrophoresis by Hjerten et al. [S. Hjerten, K. Elenbring, F. Kilar, J.-L. Liao, A. J. C. Chen, C. J. Siebert and M.-D. Zhu, J. Chromatogr., 403 (1987) 47; S. Hjerten, J. Chromatogr., 347 91985) 191] with the aim of elimination of electroosmotic flow and of the avoidance of adsorption of proteins. By this procedure, a non-crosslinked monolayer of polyacrylamide is said to be bonded to the capillary surface.
Already in 1983 Hjerten also filled polyacrylamide gel into glass capillaries of 0.15 mm inner diameter [S. Hjerten, J. Chromatogr., 270 (1983) 1] and could successfully apply such capillaries for the separation of proteins [S. Hjerten, M.-D Zhu, J. Chromatogr., 327 (1985) 157; S. Hjerten K. Elenbring, F. Kilar, J.-L. Liao, A. J. C. Chen, C. J. Siebert and M.-D. Zhu J. Chromatogr., 403 (1987) 47]. In various publications since 1986 Cohen and Karger have shown electrophoretic separations of both classes of biomolecules, proteins and oligonucleotides, which were achieved in gel filled fused silica capillaries. The authors concluded from their experience with the generation and application of such gel capillaries that the gel should be chemically bonded to the capillary surface by treatment of the surface with bifunctional reagents such as 3-methacryloxypropyltrimethoxysilane before the final gel is generated in the capillary [A. S. Cohen and B. L. Karger, J. Chromatogr., 397 (1987) 409; B. L. Karger and A. S. Cohen, Northeastern University, U.S. Pat., U.S. Pat. No. 94 865 706, Oct. 21, 1986; B. L. Karger and A. S. Cohen, Northeastern University, Eur. Pat., EP 324 539 A2, May 1, 1989]. Gel electrophoresis in capillaries can also successfully be performed without pretreatment of the capillary surface [S. Hjerten, J. Chromatogr., 270 (1983) 1]. According to our own experiments, a solution of the acrylamide/bisacylamide mixture can simply be filled into fused-silica capillaries and polymerized either by chemical reagents such as peroxide sulfate or by .gamma.-rays [J. A. Lux, H.- F. Yin and G. Schomburg, HRC, 13 (1990) 436]. Polymerization of gels on slabs by radiation has been described before [E. I. DuPont de Nemours and Company, Wilmington, Delaware 1989, Eur. Pat., EP OS 0159 694 A2, Oct. 30, 1985]. Gels polymerized in capillaries which are free of bubbles could easily be obtained with .gamma.-radiation without prior surface treatment [FIG. 1 A; J. A. Lux, H.-F. Yin and G. Schomburg, HRC 13 (1990) 436]. Electropherograms achieved by the authors with gel capillaries which have been manufactured without and with pretreatment of the fused silica surface are compared with regard to resolution and especially peak symmetry [FIG. 2A]. By other authors, gel capillaries obtained without surface pretreatment were found to be insufficiently stable, i.e. their performance was found to decrease rapidly during usage. According to our experience capillaries could be used for as much as 200 measurements without appreciable decrease in performance, provided the gel formation during column production was properly controlled. Only a small section of the gel is likely to be slowly extruded out of the capillary by influence of the electroosmotic flow arising on the untreated surface. By this the separation performance decreases slightly. By occasional cutting off the very first few millimeters of the capillary the separation performance can be restored, however. Decreased life-times of gel capillaries caused by such effects have also been reported before (H. Swerdlow and R. Gesteland, Nucleic Acids Research, 18 (1990) 1415].
It appeared to be difficult for other research groups to produce suitable gel capillaries following the procedures described by Cohen and Karger [B. L. Karger and A. S. Cohen, Northeastern University, U.S. Pat., U.S. Pat. No. 94 865 706, Ovy. 21, 1986, B. L. Karger and A. S. Cohen, Northeastern University, Eur. Pat., EP 324 539 A2, May 1, 1989]. Cohen and Karger's method is based on the utilization of a bifunctional compound, for example the well known 3-methacryloxypropyltrimethoxysilane, for surface pretreatment. In this reagent one part of this molecule (i.e. that which contains the three methoxy groups) is intended to effect the chemical bonding to the fused-silica surface while the double bond in the other part of the molecule is available for subsequent copolymerization with acrylamide/bisacrylamide during the formation of the gel. In this way the gel becomes bonded to the fused-silica. According to our trials the unavoidable shrinking of the acrylamide/bisacrylamide mixture during the gel formation by polymerization must take place without any chemical bonding of the gel to the surface, less bubbles arise. By the chemical bonding, sections of the gel under formation are fixed to the surface and prevented from moving unrestrictedly within the capillary during the process of polymerization. The appearance of equidistantly spaced bubbles in the capillary can thus be explained. It has also been tried to generate bubble-free gels, by application of high pressures of up to 400 bar during gel formation generated by means of a HPLC pump [P. F. Bente and J. Myerson, Hewlett-Packard Co., Eur. Pat., EP 272 925 A2, June 29, 1988; U.S. Pat., U.S. Pat. No. 4,810,456, Dec. 24, 1986]. Also, linear hydrophilic polymers such as PEG have been added to the acrylamide/bisacrylamide solution to facilitate the shrinking process of the gel.
Beyond the procedure of gel capillary production described by Cohen and Karger in their patents, we successfully included an additional step of surface pretreatment before the actual gel formation in the capillary is effected. In this step a layer of linear polyacrylamide is generated by starting a polymerization of acrylamide from the vinyl groups which are present on the surface after the pretreatment with the 3-methylacryloxypropyltrimethoxysilane.